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A homogenous luminescent proximity assay for 14-3-3 interactions with both phosphorylated and nonphosphorylated client peptides.

Du Y, Khuri FR, Fu H - Curr Chem Genomics (2008)

Bottom Line: The 14-3-3 proteins are a family of dimeric eukaryotic proteins that mediate both phosphorylation-dependent and -independent protein-protein interactions.Through these interactions, 14-3-3 proteins participate in the regulation of a wide range of cellular processes, including cell proliferation, cell cycle progression, and apoptosis.Because of their fundamental importance, 14-3-3 proteins have also been implicated in a variety of diseases, including cancer and neurodegenerative disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
The 14-3-3 proteins are a family of dimeric eukaryotic proteins that mediate both phosphorylation-dependent and -independent protein-protein interactions. Through these interactions, 14-3-3 proteins participate in the regulation of a wide range of cellular processes, including cell proliferation, cell cycle progression, and apoptosis. Because of their fundamental importance, 14-3-3 proteins have also been implicated in a variety of diseases, including cancer and neurodegenerative disorders. In order to monitor 14-3-3/client protein interactions for the discovery of small molecule 14-3-3 modulators, we have designed and optimized 14-3-3 protein binding assays based on the amplified luminescent proximity homogeneous assay (AlphaScreen) technology. Using the interaction of 14-3-3 with a phosphorylated Raf-1 peptide and a nonphosphorylated R18 peptide as model systems, we have established homogenous "add-and-measure" high-throughput screening assays. Both assays achieved robust performance with S/B ratios above 7 and Z' factors above 0.7. Application of the known antagonistic peptides in our studies further validated the assay for screening of chemical compound libraries to identify small molecules that can modulate 14-3-3 protein-protein interactions.

No MeSH data available.


Related in: MedlinePlus

Evaluation of the DMSO tolerance and stability of the 14-3-3 AlphaScreen assay. (A) Effect of DMSO on Alpha signal. The biotin-14-3-3γ (5 nM) and GST-R18 (15 nM) were incubated with increasing amounts of DMSO, and reactions were carried out as described in the legend to Fig. (4). Results were recorded after incubation with donor and acceptor beads for 1.5 hr. (B) Stability of the 14-3-3 AlphaScreen assay as evaluated in R18 competition assay. Experiments were carried out as described in Fig. (4) except that results were recorded after incubation for 1.5 hr or 16 hr. Dose-response data with the untagged R18 peptide antagonist were expressed as the percentage of maximal Alpha signal control that was obtained from samples in the absence of inhibitor. Data are expressed as means ± SD from triplicate determinations.
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Figure 6: Evaluation of the DMSO tolerance and stability of the 14-3-3 AlphaScreen assay. (A) Effect of DMSO on Alpha signal. The biotin-14-3-3γ (5 nM) and GST-R18 (15 nM) were incubated with increasing amounts of DMSO, and reactions were carried out as described in the legend to Fig. (4). Results were recorded after incubation with donor and acceptor beads for 1.5 hr. (B) Stability of the 14-3-3 AlphaScreen assay as evaluated in R18 competition assay. Experiments were carried out as described in Fig. (4) except that results were recorded after incubation for 1.5 hr or 16 hr. Dose-response data with the untagged R18 peptide antagonist were expressed as the percentage of maximal Alpha signal control that was obtained from samples in the absence of inhibitor. Data are expressed as means ± SD from triplicate determinations.

Mentions: First, the dimethyl sulfoxide (DMSO) tolerance level of the assay was evaluated. DMSO is a common solvent used in dissolving chemical compounds. To determine the effect of DMSO on the AlphaScreen based 14-3-3 assay, experiments were performed in the presence of varying amounts of DMSO. As shown in Fig. (6A), the Alpha signals for the binding of biotin-14-3-3γ (5 nM) to GST-R18 (15 nM) were not significantly altered in the presence of up to 5% DMSO. Then, we determined the assay stability by performing competition experiments and recording the AlphaScreen luminescence signal over time. As shown in Fig. (6B), the addition of untagged R18 peptide to the reaction containing biotin-14-3-3γ, GST-R18, and the donor/acceptor beads, decreased the Alpha luminescence signals in a dose-dependent manner. No significant difference was noticed when the result was recorded either at 1.5 hr or 16 hr after mixing the reaction components. Similar IC50 values were obtained with these two time points (28.0 ± 1.1 nM for 1.5 hr and 33.6 ± 1.1 nM for 16 hr). These results suggest that the AlphaScreen assay for HTS of 14-3-3 inhibitors is stable over a time period of at least 16 hr, which allows the reading of more than 120 microplates (384-well) with the Envision plate reader. Finally, we evaluated parameters for HTS performance and assay consistency by testing well-to-well and plate-to-plate variation of the binding assay in a total volume of 20 μl. Each reaction contained 5 nM of biotin-14-3-3 and 15 nM of GST-R18 as in Fig. (3). The Z’ factor and S/B ratio of reactions from ten individual plates were recorded and compared (Figs.7A and 7B). Among these 10 plates, the average value for Z’ was 0.73 with a standard deviation of 0.06 and the average S/B ratio was 10.6 with a standard deviation of 2.4, indicating the robustness and suitability of this assay for automation. We further evaluated the assay consistency by performing the R18 peptide competition assay with the biotin-14-3-3γ/GST-R18 interaction pair on different days. As shown in Fig. (7C), the dose-response curves obtained from measurements in two different plates at two different days gave rise to similar IC50 values ( 27.3 nM and 43.2 nM, respectively), demonstrating high consistency of the AlphaScreen assays.


A homogenous luminescent proximity assay for 14-3-3 interactions with both phosphorylated and nonphosphorylated client peptides.

Du Y, Khuri FR, Fu H - Curr Chem Genomics (2008)

Evaluation of the DMSO tolerance and stability of the 14-3-3 AlphaScreen assay. (A) Effect of DMSO on Alpha signal. The biotin-14-3-3γ (5 nM) and GST-R18 (15 nM) were incubated with increasing amounts of DMSO, and reactions were carried out as described in the legend to Fig. (4). Results were recorded after incubation with donor and acceptor beads for 1.5 hr. (B) Stability of the 14-3-3 AlphaScreen assay as evaluated in R18 competition assay. Experiments were carried out as described in Fig. (4) except that results were recorded after incubation for 1.5 hr or 16 hr. Dose-response data with the untagged R18 peptide antagonist were expressed as the percentage of maximal Alpha signal control that was obtained from samples in the absence of inhibitor. Data are expressed as means ± SD from triplicate determinations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803432&req=5

Figure 6: Evaluation of the DMSO tolerance and stability of the 14-3-3 AlphaScreen assay. (A) Effect of DMSO on Alpha signal. The biotin-14-3-3γ (5 nM) and GST-R18 (15 nM) were incubated with increasing amounts of DMSO, and reactions were carried out as described in the legend to Fig. (4). Results were recorded after incubation with donor and acceptor beads for 1.5 hr. (B) Stability of the 14-3-3 AlphaScreen assay as evaluated in R18 competition assay. Experiments were carried out as described in Fig. (4) except that results were recorded after incubation for 1.5 hr or 16 hr. Dose-response data with the untagged R18 peptide antagonist were expressed as the percentage of maximal Alpha signal control that was obtained from samples in the absence of inhibitor. Data are expressed as means ± SD from triplicate determinations.
Mentions: First, the dimethyl sulfoxide (DMSO) tolerance level of the assay was evaluated. DMSO is a common solvent used in dissolving chemical compounds. To determine the effect of DMSO on the AlphaScreen based 14-3-3 assay, experiments were performed in the presence of varying amounts of DMSO. As shown in Fig. (6A), the Alpha signals for the binding of biotin-14-3-3γ (5 nM) to GST-R18 (15 nM) were not significantly altered in the presence of up to 5% DMSO. Then, we determined the assay stability by performing competition experiments and recording the AlphaScreen luminescence signal over time. As shown in Fig. (6B), the addition of untagged R18 peptide to the reaction containing biotin-14-3-3γ, GST-R18, and the donor/acceptor beads, decreased the Alpha luminescence signals in a dose-dependent manner. No significant difference was noticed when the result was recorded either at 1.5 hr or 16 hr after mixing the reaction components. Similar IC50 values were obtained with these two time points (28.0 ± 1.1 nM for 1.5 hr and 33.6 ± 1.1 nM for 16 hr). These results suggest that the AlphaScreen assay for HTS of 14-3-3 inhibitors is stable over a time period of at least 16 hr, which allows the reading of more than 120 microplates (384-well) with the Envision plate reader. Finally, we evaluated parameters for HTS performance and assay consistency by testing well-to-well and plate-to-plate variation of the binding assay in a total volume of 20 μl. Each reaction contained 5 nM of biotin-14-3-3 and 15 nM of GST-R18 as in Fig. (3). The Z’ factor and S/B ratio of reactions from ten individual plates were recorded and compared (Figs.7A and 7B). Among these 10 plates, the average value for Z’ was 0.73 with a standard deviation of 0.06 and the average S/B ratio was 10.6 with a standard deviation of 2.4, indicating the robustness and suitability of this assay for automation. We further evaluated the assay consistency by performing the R18 peptide competition assay with the biotin-14-3-3γ/GST-R18 interaction pair on different days. As shown in Fig. (7C), the dose-response curves obtained from measurements in two different plates at two different days gave rise to similar IC50 values ( 27.3 nM and 43.2 nM, respectively), demonstrating high consistency of the AlphaScreen assays.

Bottom Line: The 14-3-3 proteins are a family of dimeric eukaryotic proteins that mediate both phosphorylation-dependent and -independent protein-protein interactions.Through these interactions, 14-3-3 proteins participate in the regulation of a wide range of cellular processes, including cell proliferation, cell cycle progression, and apoptosis.Because of their fundamental importance, 14-3-3 proteins have also been implicated in a variety of diseases, including cancer and neurodegenerative disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
The 14-3-3 proteins are a family of dimeric eukaryotic proteins that mediate both phosphorylation-dependent and -independent protein-protein interactions. Through these interactions, 14-3-3 proteins participate in the regulation of a wide range of cellular processes, including cell proliferation, cell cycle progression, and apoptosis. Because of their fundamental importance, 14-3-3 proteins have also been implicated in a variety of diseases, including cancer and neurodegenerative disorders. In order to monitor 14-3-3/client protein interactions for the discovery of small molecule 14-3-3 modulators, we have designed and optimized 14-3-3 protein binding assays based on the amplified luminescent proximity homogeneous assay (AlphaScreen) technology. Using the interaction of 14-3-3 with a phosphorylated Raf-1 peptide and a nonphosphorylated R18 peptide as model systems, we have established homogenous "add-and-measure" high-throughput screening assays. Both assays achieved robust performance with S/B ratios above 7 and Z' factors above 0.7. Application of the known antagonistic peptides in our studies further validated the assay for screening of chemical compound libraries to identify small molecules that can modulate 14-3-3 protein-protein interactions.

No MeSH data available.


Related in: MedlinePlus